Leishmaniasis vectors in the environment of treated leishmaniasis cases in Spain

Abstract Transmission of leishmaniasis in endemic areas is characterized by microfocality related to the presence of the vector. Most entomological studies in southwestern Europe have focused on sylvatic areas and town outskirts, very few have sampled town or urban centres, and no survey has investigated inside households. The aim of this study was to determine the sand fly species diversity and vector density in the surroundings of human leishmaniasis cases compared with environments in which there was no association. Sand flies were captured in 26 households associated with recently treated leishmaniasis patients, 15 neighbouring houses without associated cases, and in others environments. Overall 7495 sand flies belonging to six species were captured. The highest sand fly density was found in farmhouses where there is a great availability of blood sources and breeding sites. In the environment of human leishmaniasis cases, Sergentomyia minuta was the most prevalent species followed by Phlebotomus perniciosus. Nevertheless, lower Leishmania infantum infection rates and lower intensity of infection were detected in S. minuta sand flies than in P. perniciosus. The density of P. perniciosus in households with recently treated leishmaniasis patients varies between 0 and 108 sand flies per light trap/night, with the maximum values corresponding to farmhouses. This species appears to be adapted to both indoors and outdoors domestic biotopes, including urban households.


INTRODUCTION
Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania that is transmitted through the bite of infected female sand flies. The presence of sand fly vectors is considered the main risk for the occurrence of leishmaniasis in an area. Non-vectorial transmission routes such as blood transfusions, needle reuse or sexual and vertical transmission have barely been reported in endemic areas, but some studies show evidence of increased risk posed by these alternative routes (Jiménez-Marco et al., 2016;Martín-Sánchez, Torres-Medina, et al., 2020;Morillas-Márquez et al., 2002;Pineda et al., 2001Pineda et al., , 2002. Vertical transmission appears to play an important role in the spread of leishmaniasis in prolific animal hosts (Martín-Sánchez, Torres-Medina, et al., 2020). Leishmaniasis is caused by Leishmania infantum in southwestern Europe, where human incidence is low despite the high prevalence found in dogs, its main domestic reservoir. Studies on risk factors for human infection with L. infantum have yielded conflicting results, but there is the generalized concept that owners of infected dogs and household members could be at high risk of infection, at least in the Americas (Belo et al., 2013).

Leishmania infantum infection in humans leads to clinical disease in
only a fraction of all those infected (Aliaga et al., 2019;Pérez-Cutillas et al., 2015;Riera et al., 2008), but the role of infected people without clinical signs in the epidemiological cycle is not fully elucidated ).
An increasing number of reports suggest that leishmaniasis affects many mammalian species in Europe, both domestic animals and wildlife, as recently reviewed by Cardoso et al. (2021). Wildlife is a major source of infection and interactions with domestic cycles can occur; therefore, understanding the role that wildlife plays in the epidemiology of leishmaniasis will help design measures to reduce prevalence as potential mitigation strategies.
Human intervention in many areas leads to a change in the Leishmania transmission cycle by increasing exposure to infected vectors and susceptible reservoirs, or facilitating the interaction between wild and domestic transmission cycles. Different factors related to the urbanization and changes in land use have been involved in the re-emergence of leishmaniasis in Madrid area, favouring the parasite transmission among Iberian hares and to a lesser extent wild rabbits of the green areas surrounding the municipalities affected by the outbreak (Molina et al., 2012).  (Alcover et al., 2014;Barón et al., 2011;Branco et al., 2013;Cotteaux-Lautard et al., 2016;Gálvez et al., 2010;González et al., 2021;Izri et al., 1992;Maia et al., 2013;Martín-Sánchez et al., 1994;Morillas Márquez et al., 1983;Muñoz et al., 2021;Sáez et al., 2018). Phlebotomus longicuspis presence in southern Spain has been ruled out (Martín-Sánchez et al., 2000;Pesson et al., 2004).
Changes in sand fly distribution are essential to determine the potential expansion of leishmaniasis risk areas (Ballart et al., 2012;Díaz-Sáez et al., 2021;Maroli et al., 2008). In addition, changes in sand fly distribution at the local level could condition the patchy distribution of human leishmaniasis cases in endemic areas. Updated data on vector species at peridomestic level, both intra and peridomiciliary, are necessary for a good understanding of the L. infantum transmission dynamics. Therefore, our objective was to determine the sand fly species diversity and vector density in the surroundings of human leishmaniasis cases compared with others environments in which there was no association with human leishmaniasis cases.

Study area
The present study was carried out in Granada province (Spain), in the southeast of the Iberian Peninsula (Geographical coordinates of the

Study design
Leishmaniasis is a notifiable disease in Andalusia.

Sand fly collection and morphological identification
Sand flies were captured using CDC light traps and sticky traps (21 × 29.5 cm paper sheets covered in castor oil) in the environment of (1) 24 recent human leishmaniasis cases occurred between 2015 and 2016, (2) six cryptic leishmaniasis cases found among blood donors, Nine Hundred Forty-five sticky traps (total surface 117 m 2 ) were placed in dwellings/farms walls and holes, and spread across the town or district. Individuals were taken with care from the surface of sticky papers using a brush soaked in 96 • alcohol and subsequently stored in 70% alcohol. Later, the entire specimens were placed in Marc André solution and heated to boiling point, and finally mounted on slides under a coverslip using Berlese solution.
Morphological identification was performed using taxonomic keys and were mainly based on the observation of male genitalia and female spermathecae.

Determination of parasite loads by qPCR
MasterPure DNA Purification Kit (Epicentre, Madison, WI, USA) was used for DNA extraction from female sand flies. DNA purification protocols provided by the manufacturer were used. Briefly, each sample was disrupted using a small pistil, protein was removed using proteinase K and the DNA was concentrated using isopropanol precipitation. DNA quality and quantity were determined spectrophotometrically (Nanodrop 2000c, Thermo Scientific). Extraction controls were used to ensure that the DNA was not cross-contaminated: the extraction process was simultaneously applied to test-tubes containing sterile water as well as to the biological samples. The extracted DNA was kept at −20 • C. Separately, DNA was also extracted from cultured

Data analysis
Density (number of specimens per trap and night when using CDC light traps; number of specimens per square meter of trap for sticky traps) and relative abundance (number of specimens of a given species over the total number of captured sand flies expressed as percentage) were calculated for all species identified. Software package IBM SPSS Statistics version 21.0 was used for the statistical analysis.

RESULTS
We studied the household and family environment of 24 leishmania- In the city of Granada, eight dwellings were investigated: seven associated with leishmaniasis cases and one no-case. The sand fly densities of subgenus Larroussius species, mainly P. perniciosus, captured inside and outside these households according to its location in Granada city or in the towns of the province are presented in Table 3.
Using logistic regression, no differences were detected between the densities of sand flies of Larroussius subgenus captured in dwellings located in towns and those located in Granada city (p > .299  (Alcover et al., 2014;Ballart et al., 2012;Barón et al., 2011;Gálvez et al., 2010;Prudhomme et al., 2015;Risueño et al., 2017). Most of these entomological surveys focus primarily on sampling between towns or at town limits, mainly on embankments although also farms and cottage houses. Few studies have performed surveys in towns and none inside patient households. Of the three potential vectors of L. infantum in southwestern Europe, P. perniciosus is the main cause of transmission due to its wider distribution and density. The presence of P. perniciosus and its density on embankments is greater between towns than in other locations such as town boundaries or town centres (Barón et al., 2011;Gálvez et al., 2010). However, when the characteristics of the sampling sites are not as homogeneous as the embankments and walls, the highest density is reached in biotopes that include domestic animal shelters, which tend to be more frequent in peridomestic environments located at town limits (Branco et al., 2013;Bravo-Barriga et al., 2016;Maia et al., 2013). Phlebotomus perniciosus density has been also assessed in periurban residential estates showing widely variable density data that are greater in adjacent non-urbanized sites (Muñoz et al., 2021) and in peri-urban green areas, as in the focus of leishmaniasis in Madrid where it was the only vector and showed high densities and infection rates (González et al., 2017). To the best of our knowledge, this is the only study in which sand flies captures are made in European households associated with leishmaniasis cases due to L. infantum.

Other similar surveys have been carried out in India and Nepal, where
Leishmania donovani is endemic (Picado et al., 2010); and in L. tropica Five sand fly species commonly found in southwestern Europe (P. perniciosus, P. papatasi, P. ariasi, P. sergenti and S. minuta) and the frequently overlooked, P. langeroni were found in the biotopes studied in the present study (Barón et al, 2011;Morillas Márquez et al., 1983;Sáez et al., 2018). Phlebotomus perniciosus was the most abundant and densest Larroussius species in households with recently treated leishmaniasis patients as in the other three environments, while P. ariasi abundance and density were generally low, in agreement with the preference of this species for humid or sub-humid areas (Ballart et al., 2012) as opposed to the semi-arid or arid character of the sampled areas.
Despite this fact, P. ariasi covers a wide geographic range in the western Mediterranean showing a high intra-specific diversity (Franco et al., 2010). Phlebotomus langeroni is associated with the existence of rabbit burrows in immediate surrounding areas of the capture sites, a necessary condition for the presence of this species .
The very low density of this sand fly species in domestic environments seems to indicate that although P. langeroni is a competent vector, its involvement in transmission in this environment is negligible and would be limited to the sylvatic transmission cycle.
The highest sand fly density was found in farmhouses where there is a great availability of vertebrates as blood sources for females to mature the eggs, abundant organic matter to find breeding sites and resting sites for adult sand flies to shelter during the day.
Phlebotomus perniciosus is an opportunistic biter of a wide range of hosts, including humans (Bravo-Barriga et al., 2016;González et al., 2017), and farmhouses show optimal suitability for this vector, reaching in these microenvironments the highest values of relative abundance.
Sergentomyia minuta was the most prevalent species in the study overall and in the environment of leishmaniasis cases, similar to other surveys we have carried out in peridomestic and sylvatic biotopes in the province (Barón et al., 2011 Leishmaniasis due to L. infantum is classically considered a rural zoonotic disease; however, and according to our results, P. perniciosus, the primary vector of L. infantum, can also be found in urban areas, both indoors and outdoors. Phlebotomus perniciosus also breeds in these urban peridomestic and domestic environments with enough organic matter to feed larvae. Females need blood meals, and like males, use plant as their main sources of energy and water. The existence of gardens and parterres in urban contexts is widespread in cities from southwestern Europe, and pots are common in houses, being able to provide water and energy to adults and serving as a habitat for larvae.
In conclusion, the highest sand fly density was found in farmhouses, both associated and not associated with leishmaniasis cases, where there is a great availability of blood sources and breeding sites. Houses with associated cases have a higher sand fly density than those without associated cases, although without statistical significance due to the sample size. In the environment of human leishmaniasis cases, S.
minuta is the most prevalent species but lacks the high L. infantum transmission capacity of P. perniciosus, the second most abundant and densest species. This species appears to be adapted to both indoors and outdoors domestic biotopes, including urban households.

CONFLICT OF INTEREST
The authors declare no competing interests.

ETHICS STATEMENT
The procedure was approved by the Ethics Committee of the University of Granada.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.